It has been proposed that DNA methylation is involved in the mechanism of X inactivation, the process by which equivalence of levels of X-linked gene products is achieved in female (XX) and male (XY) mammals. In this study, Southern blots of female and male DNA digested with methylation-sensitive restriction endonucleases and hybridized to various portions of the cloned mouse hprt gene were compared, and sites within the mouse hprt gene were identified that are differentially methylated in female and male cells. The extent to which these sites are methylated when carried on the active and inactive X chromosomes was directly determined in a similar analysis of DNA from clonal cell lines established from a female embryo derived from a mating of two species of mouse, Mus musculus and Mus caroli. The results revealed two regions of differential methylation in the mouse hprt gene. One region, in the first intron of the gene, includes four sites that are completely unmethylated when carried on the active X and extensively methylated when carried on the inactive X. These same sites are extensively demethylated in hprt genes reactivated either spontaneously or after 5-azacytidine treatment. The second region includes several sites in the 3' 20kilobases of the gene extending from exon 3 to exon 9 that show the converse pattern; i.e., they are completely methylated when carried on the active X and completely unmethylated when carried on the inactive X. At least one of these sites does not become methylated after reactivation of the gene. The results of this study, together with the results of previous studies by others of the human hprt gene, indicate that these regions of differential methylation on the active and inactive X are conserved between mammalian species. Furthermore, the data described here are consistent with the idea that at least the sites in the 5' region of the gene play a role in the X inactivation phenomenon and regulation of expression of the mouse hprt gene.
Human embryonic stem cells (hESC) are stem cells capable of differentiating into cells representative of the three primary embryonic germ layers. There has been considerable interest in understanding the mechanisms regulating stem cell pluripotency, which will ultimately lead to development of more efficient methods to derive and culture hESC. In particular, Oct4, Sox2 and Nanog are transcription factors known to be important in maintenance of hESC. However, many of the downstream targets of these transcription factors are not well characterized. Furthermore, it remains unknown whether additional novel stem cell factors are involved in the establishment and maintenance of the stem cell state.Here we show that a novel gene, L1TD1 (also known as FLJ10884 or ECAT11), is abundantly expressed in undifferentiated hESC. Differentiation of hESC via embryoid body (EB) formation or BMP4 treatment results in the rapid down-regulation of L1TD1 expression. Furthermore, populations of undifferentiated and differentiated hESC were sorted using the stem cell markers SSEA4 and TRA160. Our results show that L1TD1 is enriched in the SSEA4-positive or TRA160-positive population of hESC. Using chromatin immunoprecipitation we found enriched association of Nanog to the predicted promoter region of L1TD1. Furthermore, siRNA-mediated knockdown of Nanog in hESC also resulted in downregulation of L1TD1 expression. Finally, using luciferase reporter assay we demonstrated that Nanog can activate the L1TD1 upstream promoter region. Altogether, these results provide evidence that L1TD1 is a downstream target of Nanog.Taken together, our results suggest that L1TD1 is a downstream target of Nanog and represents a useful marker for identifying undifferentiated hESC.
Abstract We have investigated the effects of testicular exposure to different doses of Co 60 radiation on sperm morphology in F‐344 rats. The results indicate that from 150rad to 500 rad gamma irradiation causes statistically significant, dose‐related increases in 1) the percent of morphologically aberrant sperm and 2) the frequency of tailless sperm. Both of these effects were detectable in sperm which were derived from treated spermatid, spermatocytes, and spermatogonial cells. These data indicate that the development of a sperm morphology assay in rats is feasible.
Abstract The signaling mechanisms controlling somatic cell reprogramming are not fully understood. In this study, we report a novel role for mitochondrial Akt1 signaling that enhanced somatic cell reprogramming efficiency. The role of mitochondrial Akt1 in somatic cell reprogramming was investigated by transducing fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, c-Myc) in conjunction with Mito-Akt1, Mito-dnAkt1, or control virus. Mito-Akt1 enhanced reprogramming efficiency whereas Mito-dnAkt1 inhibited reprogramming. The resulting iPSCs formed embryoid bodies in vitro and teratomas in vivo . Moreover, Oct4 and Nanog promoter methylation was reduced in the iPSCs generated in the presence of Mito-Akt1. Akt1 was activated and translocated into mitochondria after growth factor stimulation in embryonic stem cells (ESCs). To study the effect of mitochondrial Akt in ESCs, a mitochondria-targeting constitutively active Akt1 (Mito-Akt1) was expressed in ESCs. Gene expression profiling showed upregulation of genes that promote stem cell proliferation and survival and down-regulation of genes that promote differentiation. Analysis of cellular respiration indicated similar metabolic profile in the resulting iPSCs and ESCs, suggesting comparable bioenergetics. These findings showed that activation of mitochondrial Akt1 signaling was required during somatic cell reprogramming.
